Clearance of intracellular Klebsiella pneumoniae infection usinggentamicin-loaded nanoparticles

Jiang, L., Greene, M. K., Insua, J. L., Sa-Pessoa, J., Small, D. M., Smyth, P., McCann, A., Cogo, F.,Bengoechea, J. A., Taggart, C. C., & Scott, C. J. (2018). Clearance of intracellular Klebsiella pneumoniaeinfection using gentamicin-loaded nanoparticles. Journal of Controlled Release, 279, 316.https://doi.org/10.1016/j.jconrel.2018.04.040

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Download date:26. Feb. 2022

Accepted Manuscript

Clearance of intracellular Klebsiella pneumoniae infection usinggentamicin-loaded nanoparticles

Lai Jiang, Michelle K. Greene, Jose Luis Insua, Joana Sa Pessoa,Donna M. Small, Peter Smyth, Aidan McCann, Francesco Cogo,Jose A. Bengoechea, Clifford C. Taggart, Christopher J. Scott

PII: S0168-3659(18)30224-4DOI: doi:10.1016/j.jconrel.2018.04.040Reference: COREL 9267

To appear in: Journal of Controlled Release

Received date: 8 January 2018Revised date: 16 April 2018Accepted date: 20 April 2018

Please cite this article as: Lai Jiang, Michelle K. Greene, Jose Luis Insua, Joana Sa Pessoa,Donna M. Small, Peter Smyth, Aidan McCann, Francesco Cogo, Jose A. Bengoechea,Clifford C. Taggart, Christopher J. Scott , Clearance of intracellular Klebsiella pneumoniaeinfection using gentamicin-loaded nanoparticles. The address for the corresponding authorwas captured as affiliation for all authors. Please check if appropriate. Corel(2018),doi:10.1016/j.jconrel.2018.04.040

This is a PDF file of an unedited manuscript that has been accepted for publication. Asa service to our customers we are providing this early version of the manuscript. Themanuscript will undergo copyediting, typesetting, and review of the resulting proof beforeit is published in its final form. Please note that during the production process errors maybe discovered which could affect the content, and all legal disclaimers that apply to thejournal pertain.

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Clearance of intracellular Klebsiella pneumoniae

infection using gentamicin-loaded nanoparticles Lai Jianga, Michelle K. Greenec, Jose Luis Insuaa, Joana Sa Pessoaa, Donna M. Smalla, Peter

Smythc, Aidan McCannc, Francesco Cogoc, Jose A. Bengoecheaa, Clifford C. Taggarta and Christopher J. Scottb,*. aCentre for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, Northern Ireland, UK bCentre for Cancer Research and Cell Biology, School of Medicine, Dentistry and

Biomedical Sciences, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, Northern Ireland, UK cSchool of Pharmacy, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, Northern Ireland, UK *Corresponding author. E-mail address: c.scott@qub.ac.uk (C.J. Scott).

Tel: +44(0)2890972350

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Abstract

Klebsiella pneumoniae is a foremost gram-negative pathogen that can induce life-threatening

nosocomial pulmonary infections. Although it can be phagocytosed successfully by lung

resident macrophages, this pathogen remains viable within vacuolar compartments, resulting

in chronic infection and limiting therapeutic treatment with antibiotics. In this study, we

aimed to generate and evaluate a cell-penetrant antibiotic poly(lactide-co-glycolide)

(PLGA)-based formulation that could successfully treat intracellular K. pneumoniae infection.

Screening of formulation conditions allowed the generation of high drug entrapment

nanoparticles through a water-in-oil- in-water approach. We demonstrated the therapeutic

usefulness of these gentamicin- loaded nanoparticles (GNPs), showing their ability to improve

survival and provide extended prophylactic protection towards K. pneumoniae using a

Galleria mellonella infection model. We subsequently showed that the GNPs could be

phagocytosed by K. pneumoniae infected macrophages, and significantly reduce the viability

of the intracellular bacteria without further stimulation of pro- inflammatory or pro-apoptotic

effects on the macrophages. Taken together, these results clearly show the potential to use

antibiotic loaded NPs to treat intracellular K. pneumoniae infection, reducing bacterial

viability without concomitant stimulation of inflammatory or pyroptotic pathways in the

treated cells.

Keywords: Gentamicin, PLGA, nanoparticles, Klebsiella pneumoniae, intracellular infection,

macrophage, inflammation, pyroptosis

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1. Introduction

Klebsiella pneumoniae is identified by the World Health Organisation to be of major concern

for human health [1]. It is considered one of the most important gram-negative pathogens in

nosocomial infection, frequently inducing severe pulmonary infections, particularly in elderly

patients with impaired immunological defenses [2,3]. However, it can also infect other parts

of the body including the urinary tract, lower biliary tract and surgical wound sites [4-6]. As

with other bacterial pathogens, continual use of key therapies is driving the growing

prevalence of serious antibiotic resistant strains, leading to increased concerns about present

and future treatment options [7].

Recently, it has been reported that K. pneumoniae survives intracellularly after phagocytosis

into macrophages by limiting the fusion of lysosomes with the Klebsiella containing vacuole

(KCV), creating an intracellular reservoir of infection [8]. Clinical treatment of this

intracellular pool of infection is challenging; for example, aminoglycosides, which can

successfully treat extracellular K. pneumoniae infections, have poor cellular penetration,

unsatisfactory subcellular distribution and consequently have sub-optimal activity towards

intracellular infection [9-13]. Taken together, it is clear that both new drugs and novel

delivery strategies are needed to treat this persistent and frequently lethal intracellular

pathogen.

In this current study, we have examined the ability to enhance the delivery of a clinically

relevant antibiotic into K. pneumoniae infected macrophages using an alternative

nanoformulation approach. Nanoparticle formulations are growing in popularity as potential

solutions to improve the pharmacokinetics of active pharmaceutical ingredients (API),

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alleviate systemic toxicities through drug encapsulation, and also provide controlled or

sustained release properties to extend therapeutic windows [14-18].

Nanoparticle drug systems for the treatment of K. pneumoniae have been investigated

previously. These include the application of metal nanoparticles such as gold or silver which

has inherent anti-microbial properties [19]. Antibiotic loaded nanoparticle systems have also

been examined including ceftazidime- loaded liposomes or gentamicin- loaded

chitosan/fucoidan nanoparticles to enhance pulmonary delivery of the antibiotics [20,15].

However to date, studies have not addressed the specific treatment of the intracellular

reservoir of infection. Herein we have aimed to develop a polymeric based nanoparticle with

high drug loading that would be taken up into infected macrophages by phagocytosis to

deliver the antibiotic at the site of intracellular infection; providing passive targeting of the

antibiotic into the intracellular compartments of the cells to elicit enhanced antimicrobial

effects. We demonstrate the potential effectiveness of this approach for the delivery of

gentamicin towards intracellular K. pneumoniae infection.

2. Materials and Methods

2.1 Materials

All general chemicals and reagents were supplied by Sigma-Aldrich UK, unless otherwise

specified. Gentamicin sulphate was purchased from Discovery Fine Chemicals, UK. Water

used for formulations was double distilled, HPLC grade water.

2.2 Formulation of Gentamicin- loaded PLGA Nanoparticles (GNPs)

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A water-in-oil- in-water (w/o/w) formulation strategy was adopted for the development of the

gentamicin- loaded nanoparticles. Briefly, gentamicin was dissolved in 0.5 ml of 1% aqueous

polyvinyl alcohol (PVA) solution (w/v, in 0.95% MES buffer, pH 7), followed by drop-wise

addition of 2 ml of dichloromethane (DCM) containing 20 mg of PLGA (LG 50:50, 502H,

7000-17000 Da) via a 25G needle. For rhodamine- labelled nanoparticles (RNPs), 10% (w/w)

rhodamine-B conjugated PLGA (AV011, PolySciTech, Akina, USA) was blended with

PLGA (502H). The primary emulsion was obtained by sonication at 60 watts for 12 cycles in

pulse mode (3 sec on, 2 sec off) while stirring at 1000 rpm (MS-53M multiposition stirrer,

JEIO TECH, Korea). Subsequently, 10 ml of aqueous PVA solution was poured into the

primary emulsion and sonicated for another 18 pulse sonication cycles as before. The

nanoparticle suspension was stirred for 4 hours to evaporate DCM and then washed twice by

centrifugation and resuspension cycles (at 20000 g, 20 min, 4°C) in phosphate buffered saline

(PBS). Various modifications to this formulation approach were assessed during process

optimization including varying the concentrations of PLGA and PVA, different aqueous

phases in the emulsion step and the pH of external aqueous phase as discussed in the results

section.

2.3 Characterisation of GNPs

Triplicate NP batches were diluted in PBS and characterised by Zetasizer (Nano ZS, Malvern

Instruments Ltd., UK) measurements, recording mean particle size (Zave), polydispersity

index (PDI) and zeta potential. For Scanning Electron Microscopy (SEM), small droplets (10

l, 3 mg/ml) of the final formulation of GNP and associated blank nanoparticle (BNP)

controls were dried and sputter-coated with gold on aluminum stubs and visualised (Jeol

6500 field emission gun, Japan).

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2.4 Quantification of Drug Loading in GNPs

Drug loading was calculated by analysis of residual gentamicin in the supernatants obtained

after nanoparticle precipitation. Calibration curves were prepared using known

concentrations of gentamicin dissolved in the supernatant of BNPs. In this way, any

interference was accounted for at each concentration. Based on a protocol of aminoglycoside

detection [21], 50 μl of gentamicin solution was added into a 96-well plate, followed by

addition of 50 μl of a mixture of reagent A (1 ml of 80 mg/ml O-phthaldialdehyde in 95%

ethanol) and reagent B (200 μl of 0.4 M boric acid pH 9.7, 400 μl of 2-Mercaptoethanol and

200 μl of diethyl ether). The fluorescence was measured at 360/460 nm using a fluorometer

(FLUO star Optima, BMG Labtech).

The release of gentamicin from the GNPs was assessed using 2 ml of GNPs (10 mg/ml) in

PBS buffer (at either pH 5 or pH 7), which was injected into a Slide-A-Lyzer Dialysis

Cassette 7000 MW (Thermo Scientific, UK). For comparison to free gentamicin diffusion, 2

ml of gentamicin sulfate (2 mg/ml) in PBS buffer (at either pH 5 or pH 7) was injected into

separate cassettes. The cassettes were then placed into a 30 ml reservoir of PBS buffer (at

either pH 5 or pH 7) in an incubator at 37 °C, with shaking at 120 rpm (SI50 Orbital

Incubator, Stuart Scientific, UK). At pre-determined time points, 1 ml samples were removed

from the reservoir and replaced with 1 ml fresh PBS buffer. The gentamicin content in

samples was quantified by comparison to standards containing known amounts of gentamicin

in PBS buffer (at either pH 5 or pH 7).

2.5 Bacterial Strains and Growth Conditions

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K. pneumoniae (stain 43816) was cultured on LB (Luria-Bertani) agar plates for 18 hours at

37°C. LB broth was inoculated with one bacteria colony and incubated overnight at 37°C

while shaking at 180 rpm (SI50 Orbital Incubator, Stuart Scientific, UK). The bacteria were

harvested by centrifugation (at 2500 g, 20 min, 24°C), and then diluted to a defined optical

density (A600). The colony forming units (CFU) were determined by plating serial bacterial

dilutions prepared in PBS onto agar plates and visible colonies were counted following

overnight incubation at 37°C.

2.6 in vitro Antimicrobial Activity

For planktonic K. pneumoniae assay, broth microdilution tests were performed to determine

the minimum inhibitory concentration (MIC) of GNPs against K. pneumoniae. After

overnight growth and 2.5 hrs refreshing incubation, the K. pneumoniae suspension was

adjusted to an optical density of 1.0 (A600) and diluted in LB broth to give a starting inoculum

of 5000 CFU/ml. A 100 l volume of serially diluted free gentamicin, GNPs and BNPs in LB

broth were added to a 96-well plate containing 100 l of diluted K. pneumoniae.

Concentrations of both free and nanoencapsulated gentamicin were equalised in all studies.

Polymer concentrations were also equalised when comparing BNPs and GNPs. The plates

were incubated overnight at 37°C with shaking at 120 rpm. A600 was measured and

background absorbance from the negatively controlled wells (the absorbance of LB broth or

nanoparticles only) was subtracted from all wells before analysis. The lowest concentration at

which mean A600 was zero was designated the MIC. The minimum bactericidal concentration

(MBC) was determined by the absence of growth on LB agar plates of 100 μL mixtures from

each challenged well after stipulated incubation times.

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Biofilm susceptibility assays were performed using the MBEC Assay (Innovotech,

Edmonton, Alberta, Canada). K. pneumoniae (150 μL/well, 5 x 105 CFU/mL in LB broth)

was added to an MBEC plate and incubated for 24 hours (37°C, 120 rpm) to allow biofilm

formation on the pegs. Biofilms pegs were immersed twice for 2 minutes in sterile PBS to

remove loosely adhered bacteria. Then the pegs were challenged with a range of

concentrations of either free gentamicin (0-400 μg/mL) or nanoparticle formulations in 200

μL LB broth for another 24 hours (37°C, 120 rpm). The challenge plate was then measured

for biofilm derived MIC, and the lid pegs were rinsed again and then placed in a new plate

containing fresh LB broth (recovery plate). Biofilms were disrupted by sonication for 10

minutes, and then incubated for a further 24 hours. The minimum biofilm eradication

concentration (MBEC) was designated as the lowest concentration in the recovery plate at

which there was no observable growth.

2.7 in vivo Antimicrobial Activity

The in vivo antimicrobial activity of GNPs was investigated using the in vivo Galleria

Mellonella (www.livefoodsdirect.co.uk, UK) model. In a post- infection study, LD50 K.

pneumoniae infected larvae were each treated with NP formulations or free drug (3.6 g

gentamicin equivalents) and survival percentage was monitored in the following 96 hours. In

a pre-treatment study, the same dosages of NPs and free gentamicin were given 0, 24, 48, 72,

96 and 120 hours prior to LD50 infection, and then survival percentages were monitored in

the next 120 hours. For each experiment, 10 larvae per group were studied and 20 l of

injections were administered via a 29 G needle into the last pro- legs. Hemocyte

quantification was determined from K. pneumoniae infected larvae at 1, 5, 8, 12, and 24 hrs

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post-infection. Briefly, hemolymph samples from three larvae were pooled in a

micro-centrifuge tube containing 10 l of N-phenylthiourea (Sigma). Then 50 l of trypan

blue (0.02% v/v in PBS) was added to the samples prior to enumeration by hemocytometer

after 10 minutes. Each sample was analyzed in triplicate. In a further study the residual

bacterial load from larvae was determined by isolation of the hemolymph of 10 larvae from

treatment groups at 8 hrs post- infection. The hemolymph were pooled as before in PBS and

serial dilutions of the suspensions were plated on LB agar and colonies were counted after

incubation at 37°C for 24 h.

2.8 Histopathological analysis

Histopathological analysis of the infected and treated larvae was performed through larvae

fixation in 10% formalin for 3 days at room temperature, before processing with a Leica

TP1020 processor, embedded and sectioned with Leica RM2235 microtome. The sections

were de-paraffinised in three changes of xylene (Sigma, UK) for 15 min each. The tissues

were then hydrated over an ethanol-to-water (95-70% ethanol v/v) gradient, for 5 min each.

Thereafter, the sections were washed in water for 2 min and transferred into pre-filtered

acidified Harris haematoxylin stain (Surgipath Leica Biosystems, UK) for 5 min. Then the

sections were washed with water for another 30 sec and cleared with acid alcohol for 15 sec.

After a further wash in water, the sections were immersed in aqueous eosin stain (Surgipath

Leica Biosystems, UK) for 5 min. Then the sections were dehydrated over a water-to-ethanol

gradient (70-100 % ethanol v/v), for 1 min each. Following submersion in xylene for 5 min,

one drop of DPX mountant was applied to each section and coverslips were positioned. The

slides were incubated overnight at room temperature and followed checked by a DM5500B

microscope (Leica Microsystems, Germany).

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2.9 Confocal Microscopy

For the localization of K. pneumoniae and NPs in Galleria mellonella, GFP-tagged K.

pneumoniae 43816 and RNPs were injected into larvae and incubated for 5 hrs. Section

samples were prepared as described above. After a dewax step, the sections were covered by

mounting medium with DAPI (Vector Laboratories, USA).

For the subcellular localization of K. pneumoniae and NPs in in vitro macrophage studies,

murine alveolar macrophage MH-S (ATCC CRL-2019) cell line were seeded on a 24-well

plate containing individual 12 mm circular coverslips. After infection and treatments, the

coverslip was washed with PBS twice and fixed with 4% PFA for 20 min in the dark.

Staining was carried out in 10% horse serum and 0.1% saponin in PBS. Coverslips were

washed twice in PBS and incubated for 1 hour with rat anti-Lamp-1 antibody (1D4B, Santa

Cruz Biotechnology Inc., Germany). Then the coverslips were washed again and incubated

for 45 min with PBS containing 10% horse serum, Hoechst 33342 (Invitrogen, UK) and

donkey anti-rat conjugated to Cascade blue secondary antibody (Jackson ImmunoResearch

Inc., USA). Coverslips were washed three times in PBS and once in distilled water before

mounting onto glass slides using Prolong Gold anti-fade mounting gel (Invitrogen, UK).

2.10 Assessment of Cytotoxicity

For assessment of formulation toxicity, MH-S cells were seeded at 4 x 104 per well in a

96-well white plate and treated with various concentrations of GNPs and BNPs. After

treatments, the media in each well was carefully replaced by 100 l of fresh media and 100 l

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of CellTiter Glo reagent (Promega, UK) was then added into each well. The plates were

placed on a shaker to induce cell lysis for 2 minutes and transferred to darkness for 10

minutes to stabilize the luminescent signal at room temperature. A spectrophotometer

(Synergy 2 Multi-Mode Reader, BioTek Instruments, USA) was used to measure

luminescence and cell viability was calculated as a relative percentage compared with

untreated MH-S cells.

2.11 GNPs Treatment of Infection in an Intracellular Co-Culture Model

For the intracellular co-culture model, MH-S and human monocytic THP-1 (ATCC TIB-201)

cells were separately seeded in RPMI-1640 medium (10% FCS, without antibiotics) at a

density of 3 x 105 per well in a 24-well plate 18 hours before experiments. Infections were

performed as previously described with minor modifications. Briefly, bacteria were grown in

LB broth overnight, harvested by centrifugation (2500 g, 20 min, 25°C), washed once in PBS

and adjusted to an optical density of 1.0 (A600), equating to approximately 5 x 108 CFU/mL.

Cells were infected with 100 l of bacterial suspension to give a multiplicity of infection

(MOI) of 100. The plate was centrifuged at 200 g for 5 min to synchronize the infection and

then incubated at 37°C for 1 hour. After contact, the cells were washed twice with PBS and

incubated for another 45 min with fresh RPMI-1640 (10% FCS, 100 g/ml of gentamicin).

The cells were then washed in PBS and incubated with fresh RPMI-1640 (10% FCS, 30

g/ml of gentamicin) containing various concentrations of GNPs and BNPs. For the

untreated group (free gentamicin group), the cells were incubated in RPMI-1640 (10% FCS,

30 g/ml of gentamicin) only. The inclusion of free gentamicin in the media is a standard

procedure to eliminate extracellular or cell surface bound bacteria which could contaminate

determination of intracellular infection [8]. After treatments, the cells were washed twice

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with PBS and lysed with 300 l of 0.1% saponin in PBS for 10 min at room temperature.

Serial dilutions in PBS were plated on agar plates to quantify the number of intracellular

bacteria after overnight incubation. The intracellular bacterial load is represented as CFU/

well.

2.12 Caspase Activity Assay

Treated/infected cells were washed with PBS and resuspended in 100 l of lysis buffer (25

mM HEPES, 100 mM NaCl, 2 mM EDTA, 0.1 % CHAPS, 10% sucrose, pH 7.4) before

incubation on ice for 30 mins. Samples were periodically vortexed and at the end of the

incubation, sonicated for 10 secs on ice to complete lysis. Once completed, the lysates were

clarified by centrifugation at 16000 g for 20 mins at 4°C and protein content was quantified

by BCA assay (Pierce BCA protein assay kit, Thermo, UK). Clarified lysate samples (50 g

protein) were added in triplicate to a black 96-well plate in caspase activity buffer (50 mM

HEPES, 1 mM EDTA, 1 % CHAPS, 10% sucrose, 10 mM DTT, pH 7.40) to a final volume

of 200 l per well. Caspase 1 or caspase 3 activity was measured by addition of

Ac-YVAD-AMC (ALX-260-024, Enzo Life Science, UK) or Ac-DEVD-AMC

(ALX-260-031, Enzo Life Science, UK) (final concentration, 50 M), and measurement of

increase in fluorescence over 60 mins was recorded.

For visualisation of cytoplasmic caspase-1 activity in infected and treated macrophages,

FAM-FLICA® caspase-1 probe (Immuno Chemistry Technologies, USA) was incubated

(prepared and diluted as per manufacturer’s instructions) with seeded and treated

macrophages prepared as above for 1 h. Then the media was removed and cells were washed

with PBS for 3 times. The cells were fixed and stained with Hoechst 33342. Confocal

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microscopy was carried out with a Leica TCS SP8 confocal microscope (Leica Microsystems

Ltd., UK) and images were taken with a Leica DFC350FX monochrome camera.

2.13 Statistical Analysis

Results were analysed with GraphPad Prism, version 6.0c, GraphPad Software (San Diego,

USA). G. mellonella survival shown as Kaplan-Meier plots and analysis was performed using

the log rank (Mantel-Cox) test for significance. As detailed in figure legends, t-tests and

one-way ANOVA analyses were conducted. Statistical significance critical values were

defined as *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.

3. Results and Discussion

3.1 Enhanced Formulation of Gentamicin- loaded Nanoparticles

Gentamicin is a broad-spectrum aminoglycoside antibiotic which is widely used against K.

pneumoniae. However, it is poorly cell permeable, which limits its clinical effectiveness to

treat infections caused by intracellular facultative pathogens. Notably, there is a growing

body of evidence demonstrating that K. pneumoniae survives inside macrophages in vitro and

in vivo [22], making it essential to target this intracellular population to clear infection. In this

report, we demonstrate the utility of a nanoparticle-based antibiotic delivery system in

achieving this goal.

We have previously explored the development of alginate/chitosan and PLGA nanocarriers

for the formulation of aminoglycoside antibiotics for controlled release purposes [23,24].

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With respect to PLGA-based formulations we explored an evaporation approach to generate

particles encapsulating some 22 g gentamicin per mg PLGA, bettering previous reports of

6-10 g/mg PLGA [25]. However, for this current work we re-evaluated our formulation to

further enhance drug loading. A screen of formulation parameters including polymer and

surfactant concentrations, employment of surfactant-containing aqueous phases in both

emulsification steps and pH of the aqueous phase was examined (supplementary Figure 1).

Through this approach, we determined that the use of a water-in-oil- in-water formulation

process, and the presence of PVA surfactant in the second aqueous partition at neutral pH

produced the maximum drug loading (schematic Figure 1), generating GNPs with up to 135

μg drug encapsulation per mg PLGA. Physical analysis of GNPs produced by this approach

using Dynamic Light Scattering (DLS) confirmed their diameter as 227 nm, with

monodisperse nature with a PDI of 0.162 and a zeta potential of -1.67 mV, which was further

confirmed by SEM (Figure 2).

3.2 Assessment of in vitro Antimicrobial Activity of Gentamicin- loaded Nanoparticles

The biological activity of the GNPs was assessed in comparison to free drug at identical

concentrations against planktonic K. pneumoniae (strain 43816) cultures in overnight

incubations. The MICs of free gentamicin and GNPs were determined as 1.09 g/ml and

10.94 g/ml respectively (Figure 3A). Analysis of corresponding MBCs were determined as

1.09 g/ml for the free drug and 10.94 g/ml for the GNPs (supplementary Table 1). As

expected, although the GNPs exhibited antimicrobial effects, these were reduced in

comparison to equivalent concentrations of the free drug. It was postulated that this was a

consequence of the encapsulation of the drug in the GNPs, thus preventing its immediate

exposure to the bacteria. Therefore, we conducted incubations with the bacteria over a time

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course of up to 120 hrs, observing an enhancement of MBC for the GNP to 5.47 g/ml at 96

and 120 hrs timepoints (whereas the free gentamicin MBC did not change), substantiating

this hypothesis (supplementary Table 1). This was further confirmed by analysis of the drug

release from the GNPs at pH 7.4 which showed a controlled release profile where at 120 hrs

only 33% of the drug had been released under these experimental conditions (Figure 3B).

Given that the overall strategy of the study was to examine the capability of these GNP to

deliver their antibiotic payload intracellularly within KCVs, we also examined their release

profile at pH 5.5, more representative of late endosomal lumens. Here an accelerated release

was observed (Figure 3B), where at 120 hrs over 80% of the drug was released, highlighting

the potential for this formulation to provided ‘triggered’ drug release once taken up by

infected cells.

K. pneumoniae are known to be capable of generating biofilms [26,27], and these bacteria are

more resistant to antibiotics including gentamicin [28,29]. First, the growth of

biofilm-derived planktonic bacteria was examined. The biofilm-derived planktonic bacteria

had increased tolerance to gentamicin as expected [27], where the MICs of free gentamicin

and GNPs were measured at 25 g/ml and 200 g/ml (Figure 3C). Next, we analysed the

ability of the GNPs and free drug control to inhibit growth of K. pneumoniae in 24 hrs

MBEC plate preformed peg biofilms. Following 24 hrs antibiotic treatments, recovered

bacteria were propagated establishing MBECs of free gentamicin and GNPs at 100 g/ml and

400 g/ml, respectively (Figure 3D). Although the anti-biofilm effect of GNPs was reduced

in comparison to free drug, these findings are in agreement with previous studies showing

similar activity of antibiotics released from PLGA NPs in vitro [30].

3.3 Gentamicin- loaded NPs provide longer window of protection against K. pneumoniae in

vivo

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Given the fact that a simple in vitro antimicrobial evaluation cannot show the potential of the

GNPs, we employed the Galleria mellonella larvae in vivo model to further assess their

antimicrobial activity towards K. pneumoniae infection. The G. mellonella model is widely

used for antibiotic susceptibility and pharmacokinetic investigations and has been shown to

have a positive correlation with mammalian models in determining virulence against

gram-negative bacteria [31-33]. In agreement with published results, we established an LD50

of infection at 104 CFU/larvae (Figure 4A) [34]. Next, in an initial treatment study, larvae

where challenged with 1x104 Klebsiella bacteria, and then free gentamicin solution (3.6

g/larvae), GNPs (40 g/larvae, at equivalent drug concentration), as well as non-drug

loaded NPs or PBS (10 l) sham treatment controls. The analysis of larvae survival

demonstrated that in the presence of PBS or non-drug loaded NPs as sham treatments, the

larvae succumbed to the infection by 96 h (Figure 4B). However, when treated with either

free drug or GNPs, survival was significantly enhanced. Importantly, these results

highlighted that the nanoparticle formulation was as effective as the free drug in this in vivo

model of infection. Furthermore, these results also highlighted the tolerance of the larvae to

both the BNPs and GNPs, providing confidence in their potential biocompatibility. This

suggests that in a more complex physiological microenvironment, similar levels of drug are

exposed to the infection whether from free or NP formulation.

To further understand how the GNPs protect larvae survival from K. pneumoniae infection,

we analysed the distribution of the infection and GNPs in the larvae. After infection and

treatment with RNP, we were able to discern by fluorescence microscopy that the

GFP-labelled K. pneumoniae and nanoparticles were both located within the hemocyte-rich

fat body (FB) (Supplementary Figure 2).

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Histochemical analysis of the tissue from sectioned larvae then allowed us to observe the K.

pneumoniae infected sites, which in non-drug treated larvae, became progressively melanised

with time (black arrows, Supplementary Figure 3). This is consistent with hemocyte

phagocytosis of foreign bodies and subsequent nodulization [35]. We also quantified

hemocytes and bacterial counts from these larvae, establishing that infection significantly

reduced hamocyte numbers (Figure 4C) and that bacteria where able to survive 8 h post

infection (Figure 4D), in agreement to our previous work [34]. However, it was also clear

that the gentamicin treatment (both in free or NP formulation) was able to prevent both tissue

damage (supplementary Figure 3) and reduction in hemocyte numbers (Figure 4C), whilst

concomitantly ablating residual bacteria viability (Figure 4D). This is in agreement with a

recent study in G. mellonella showing that antibiotic treatment of candida albicans could

reduce tissue damage in the larvae [36].

We hypothesized that the established controlled release properties of the GNPs may provide

an extended protective window against subsequent K. pneumoniae infection in Galleria

larvae. Larvae were treated prophylactically with either free drug or GNPs up to five days

prior to bacterial challenge, and survival monitored for a subsequent five days (Figure 5).

These experiments revealed that whilst a 24 hrs pretreatment of both free and NPs entrapped

gentamicin elicited no significant differences in survival to subsequent infection, it was clear

that with both 48 and 72 hrs pretreatments, the GNP provided significantly enhanced

protection. These results are consistent with the controlled release effect of the formulation

providing an extended protective window against infection. However, at longer 96 and 120

hrs timepoints (Figure 5E and 5F), these protective effects were lost. Taken together, these

studies in the larvae model demonstrate the therapeutic potential of the GNPs, which was not

clearly evident from the initial in vitro MIC studies. In the initial treatment model data in

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Figure 4B where the GNP and free drug were similarly effective, it could be that the drug is

rapidly released from the GNP to allow similar levels of exposure as the free drug. However,

when considered with the latter findings showing the controlled release effect, this would

suggest that the drug in its free form is rapidly excreted from the larvae, but this would

require further analysis. Interestingly in humans, gentamicin has a short half- life (40 minutes

in the lung) and is excreted rapidly through the kidneys [37], and therefore similar enhanced

therapeutic effects could be achieved clinically.

3.4 GNPs can Abrogate Intracellular K. pneumoniae Infection

Next we examined the potential of the GNPs to be taken up by K. pneumoniae infected

macrophages and to inactivate the viability of these intracellular pathogens. Recent research

has clearly shown that macrophages can act as ‘reservoirs’ for intracellular bacteria in order

to avoid clearance leading to chronic infection [22,38-40]. Based on this finding, a

macrophage-Klebsiella co-culture model was employed to assess intracellular anti-bacterial

activities of gentamicin- loaded nanoparticles in Klebsiella- infected macrophages [8]. As

professional phagocytes, we reasoned that the GNPs would be passively targeted and

engulfed by macrophages. Macrophages with GFP-labelled tagged K. pneumoniae (green)

and RNPs (red), and sub-cellular localization examined after 3 hrs by confocal microscopy.

The images in Figure 6A show that both the particles and the bacteria are internalized into the

cells and that these are contained together with LAMP1-positive KCV which live Klebsiella

prevents fusing with lysosomes as we have previously observed [8]. This co- localisation of

the particles and bacteria would suggest that drug loaded particles would have access to the

intracellular infection within the macrophages. Importantly, control experiments showed that

at these concentrations the PLGA NPs elicited no obvious toxicity towards macrophage

populations (Figure 6B). Next, we treated macrophages harboring the bacteria with the GNPs

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for 3 hrs, and upon detergent lysis of the macrophages, bacterial colony forming units were

quantified. This analysis clearly showed a significant reduction in CFUs upon treatment with

the GNPs over controls in murine alveolar macrophage (MH-S) (Figure 6C). In order to

examine a longer infection and treatment window, we used human monocytic THP1 cells,

which are more tolerant toward intracellular K. pneumoniae (18 hrs). As before, GNP

treatment effectively cleared the intracellular infection from these cells (Figure 6D).

3.5 GNPs can reduce inflammatory and pyroptotic caspase signatures in infected

macrophages

We next examined caspase activities in order to more fully appreciate the effects of the GNPs

treatment on the infected cells. The triggering of inflammasome activation by intracellular

gram-negative bacteria infection has been reported previously [33,41], but particulates can

also induce inflammatory responses and caspase activation [42]. Recent studies have shown

that intracellular infection of macrophages can activate caspase-1, which can lead to a

pyroptotic cell death [41,43-45]. In agreement we found that 4 hrs infection of K.

pneumoniae in MH-S cells generated a marked increase in caspase 1 activity in cell lysates.

Although treatment of non- infected macrophages with the GNPs also generated a small

increase in caspase 1 activity, we encouragingly found that GNP treatment of the infected

cells reduced caspase 1 activation (Figure 7A). Caspase 1 activity levels can also be

monitored through incubation of cell with a membrane-permeable FAM-FLICA® caspase-1

probe and visualized by fluorescence microscopy. Studies performed using this probe

confirmed that the GNPs were able to reduce caspase 1 activity levels in the infected MH-S

cells (Figure 7B).

Finally we also examined the activation of caspase 3. In order to detect activation of this

pro-apoptotic executor caspase we prolonged the post-infection incubation time from 3 hours

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to 24 hours, detecting a marked increase in caspase 3 activity in infected cells. Crucially,

once more we found that co-treatment of the infected cells with GNPs reduced caspase-3

activity to basal levels (Figure 7C).

4. Conclusion

We have demonstrated the effectiveness of a nanoparticle formulation strategy and generated

anti-microbial effects towards K. pneumoniae in both in vitro cultures and in vivo models.

These results support the utility of nano-antibiotic technology and our findings support the

development of GNPs for the treatment of intracellular K. pneumoniae to combat infection

and associated inflammation without compromising cellular viability. Although here we

have focused on high drug loading in our particles, further investigation into the size, charge

and other physiochemical properties of antibiotic-loaded particles may further enhance

intracellular anti-microbial effects in the future.

Funding Sources

This work was funded in part through Biotechnology and Biological Sciences Research

Council award BB/P006078/1.

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Figure 1. Schematic overview of the adopted formulation process for the preparation

of gentamicin-loaded PLGA nanoparticles (GNPs) using a water-in-oil-in-water

(W/O/W) formulation

Figure 2 A. Physical Analysis of GNPs by Dynamic Light Scattering (DLS); B.

Scanning Electron Microscope (SEM) Image of BNPs and GNPs.

Figure 3 A. Planktonic MIC determination against K. pneumoniae; B. the Release

study in PBS buffer (pH 5.5 and 7.4) at 37 ℃ over 120 hrs (Mean ± SD, n=3); C.

Biofilm-derived planktonic MIC determination (Mean ± SD, n=3); D. the MBEC

determination against 24 hrs pre-established K. pneumoniae biofilm (Mean ± SD,

n=3). Kp, K. pneumoniae; GNP, gentamicin-loaded nanoparticle; BNP, blank

nanoparticle; FG, free gentamicin.

Figure 4 A. Virulence screen of K. pneumoniae (LD50); B. The treatments against

lethal K. pneumoniae post-infection. (statistical significance analysed between all

groups [n=10 per group] using log-rank [Mantel-Cox] test, ****P<0.0001); C.

Hemocytes quantification after K. pneumoniae infection in Galleria Larvae. D.

Bacterial replication at 8 hrs after K. pneumoniae infection in Galleria Larvae.

(statistical significance analysed between all groups [n=10 per group] using one-way

ANOVA analysis in comparison to the untreated control, ****P<0.0001).

Figure 5. The Pre-Treatment Study of GNPs against K. pneumoniae in vivo.

Treatments (NPs 40 μg/Larvae [loaded gentamicin 3.6 μg/Larvae], Free Gentamicin

3.6 μg/Larvae and PBS 10 μl/Larvae) were injected into Larvae (A) 0, (B) 24, (C) 48,

(D) 72, (E) 96 and (F) 120 hours prior to the LD50 (104 CFU/Larvae) and survival

monitored up to 120 hours post-infection (Statistical significance analysed between

all groups [n=10 per group] using log-rank [Mantel-Cox] test, *P<0.05, **P<0.001,

****P<0.0001).

Figure 6 A. The sub-cellular localisation of NPs and K. pneumoniae in an infected

macrophage; B. Cell viability in NPs treated MH-S macrophages; C&D. Intracellular

bacteria quantification after NPs treatments in a MH-S (C)/THP1 (D) Macrophages &

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K. pneumoniae co-culture model (statistical significance analysed between

treatments and free gentamicin groups using one-way ANOVA, *P<0.05).

Figure 7 A. Fluorometric Caspase-1 activity assay; B. The Caspase-1 activity in

treated MH-S cells through confocal microscopy (i-Unstimulated MH-S cells; ii-GNPs

treated cells; iii-K. pneumoniae infected cells; iv-GNPs treated K. pneumoniae

infected cells, scale bar = 10 μm); C. Fluorometric Caspase-3/7 activity assay

(Statistical significance analysed between all groups using one-way ANOVA,

**P<0.01, ***P<0.001, ****P<0.0001).

Supplementary Figure 1. The optimisation strategies for gentamicin-loaded

nanoparticles formulation: the impacts of polymer concentrations, surfactant

concentration (min/max PLGA amount), different aqueous phase in 1st emulsion step

and external aqueous pH on Entrapment Efficiency% (A to E) and size (F to J).

Statistical significance analysed in comparisons with mosaic group using one-way

ANOVA, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Supplementary Figure 2. Localization of K. pneumoniae and NPs in Galleria Larvae

(arrows in each channels were scatted hemocytes [Blue], GFP-tagged K.

pneumoniae [Green] and Rhodamine NPs [Red], respectively. Scale bar = 8 μm).

Supplementary Figure 3. Histopathological analysis of NPs treated K. pneumoniae

infection in Galleria Larvae using Haemotoxylin and Eosin staining (P.I. = Post

Infection; Black arrows were melanization positions; Blue arrows were hemocytes;

Scale bar = 10 μm).

Supplementary Table 1. MBC values for GNP and free gentamicin over 5 days.

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Graphical abstract

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